(1) Field of the Invention
The present invention relates to an improved method for the reduction of cholesterol in egg materials using a cyclodextrin as a clathrating agent. In particular, the present invention relates to a method wherein a mixture of the egg material with an alkali metal hydroxide (Na or K) with heating is used to clathrate the cholesterol with the cyclodextrin and then the mixture is cooled to precipitate the clathrate which is removed.
(2) Description of Related Art
High serum cholesterol levels, high blood pressure, and abnormal electrocardiograms (ECG) are important contributing factors to heart attacks. These factors were known before the effects of smoking, obesity, and lack of exercise. One of the most consistent findings in cardiovascular studies is that high levels of plasma cholesterol are associated with atherosclerosis and thereby enhance the risk of coronary heart disease (CHD). This effect is usually mediated through the plasma low density lipoproteins (LDL), which are the most atherogenic lipoproteins.
The major causes of high serum cholesterol are due to genetic disorders, heterozygous familial hypercholesterolemia (FH), and a habitual diet high in saturated fat, calories, and cholesterol. Health experts and physicians generally agree that dietary management is the initial step in the treatment of hypercholesterolemia and hyperlipidemia. This applies even when later drug therapy is required. Changes in diet, serum cholesterol and CHD of an immigrant population have provided convincing evidence that diet plays a major role. The situation has become more complicated due to the controversial aspect of the dietary management of hypercholesterolemia and hyperlipidemia. The food industry has in recent years attempted to reduce fat and cholesterol in a wide variety of food products.
PCT W091/11114 (1991) to Oakenfull et al describes the removal of cholesterol from egg products using cyclodextrins to form a complex at temperatures between 20.degree. and 60.degree. C. and then chilling to precipitate the cholesterol-cyclodextrin complex. In this process, there is no pH adjustment of the aqueous egg material. The removal of cholesterol was less than 90%. U.S. Pat. No. 5,223,295 (1993) to Maffrand et al describes a similar process.
U.S. Pat. No. 5,063,077 (1991) to Vollbrecht et al describes the use of a solid absorbing agent to remove cholesterol from egg materials. .beta.-cyclodextrin is particularly used for this purpose. Egg granules are removed from the egg material prior to treatment in this process. After removing cholesterol, the granule and plasma fractions are recombined. There is considerable residual cholesterol in the product.
U.S. Pat. No. 5,292,546 (1994) to Cully et al describes a process wherein aqueous egg materials are treated with sodium chloride or ammonium carbonate at temperatures of less than about 10.degree. C. The problem is that with sodium chloride a residue is left in the egg material. Ammonium carbonate is removed with a vacuum which is an expensive step. Canadian Patent Application No. 2,050,031 to Cully et al describes a similar process.
PCT WO93/24022 (1993) to Hedges et al describes a process wherein the residual cyclodextrin in a food is heated at 40.degree. to 80.degree. C. with CGTase and an amylase in water to hydrolyze the cyclodextrin. This reference recognizes the problem of the cyclodextrin in the food.
U.S. Pat. No. 4,980,180 (1990) to Cully et al describes a method for removal of .beta.-cyclodextrin from egg materials using .beta.-amylases. The patent recognizes the problem of incomplete removal of the cyclodextrins at the lead of the prior art processes.
Mentink Canadian Application 2,035,156 (1991) describes in Example 1 the long heating for 5 hours of fatty acids in butter with a cyclodextrin in an aqueous solution without pH adjustment at 40.degree. C. to complex cholesterol. The fatty layer separates from the water. The cyclodextrin/cholesterol complex is treated by heating in an aqueous solution at high temperatures to separate the cholesterol.
Thus the prior art has recognized the need to remove cholesterol from egg yolk without removing or damaging those components responsible for functionality. Such a method could be used by food and pharmaceutical companies wishing to prepare egg products with reduced cholesterol content. There is a need to develop an efficient and simple process to extract the cholesterol from liquid egg yolk using .beta.-cyclodextrin, without removing or damaging the other yolk components.
Egg yolk is a complex mixture of particles held in suspension in a protein (livetin) solution. The types of particles are: yolk spheres, granules, profiles or low density lipoproteins, and myelin figures.
The granules are the major components of the yolk and represent 25% of the dry weight. They are sedimented, after diluting the yolk with an equal volume of aqueous NaCl solution (1% w/v) and centrifuging the resulting mixture at 10,000.times.g for 45 min. Granules are smaller and more numerous than the yolk spheres. The majority of granules have a diameter between 1.0 and 1.3 .mu.m. Granules contain about 70% lipovitellins, 16% phosvitin, and 12% low density lipoprotein (LDL). Granules are disassociated by a 0.6M NaCl solution and at pH 3 and 9.
Cholesterol (FIG. 1A) has a number of key functions: it is an important component of the plasma membrane of cells where it influences membrane permeability and the activity of membrane-bound enzymes; cholesterol is a precursor of isoprenoids that are involved in control of cell proliferation, bile acids and vitamin D; cholesterol is also an obligatory precursor in the synthesis of a number of steroid hormones with key roles in controlling reproduction. Over 95% of yolk cholesterol is associated with the yolk LDL. The remainder is bound to lipovitellins. Although in recent years there has been considerable discussion about the adequacy of methods of measuring yolk cholesterol, there now seems to be general agreement that eggs from commercial flocks typically contain about 213 mg per egg (USDA, Nutrient content of foods, handbook 8-1, 137-145 (1991)). Most cholesterol in yolk is in non-esterified form, although about 20% is present as cholesterol esters in hens fed normal commercial diets. Cholesterol esters, such as the stearate shown in FIG. 1B, are core components of LDL and as such appear to be more amenable to manipulation than does non-esterified cholesterol.
Concern about the cholesterol content of the human diet has led to many attempts to reduce the cholesterol content of eggs. There have been a number of different approaches, including diet alterations, drugs, breeding, solvent extraction, enzyme conversion, supercritical fluid extraction and adsorption with saponins and .beta.-cyclodextrin. The food industry has developed egg substitutes such as VIOBIN's (Viobin Corp., Monticello, Ill. 618956) desiccated defatted egg powder with the egg oil replaced by corn oil, or Fleischmann's "Egg Beaters" (Standard Brands Inc., New York, N.Y.) which contains only the egg white with other non-egg ingredients. Egg whites do not have the functional properties of egg yolk, so they are not good replacements.
Another approach is to reduce egg yolk cholesterol by processing treatments. Melnick (Melnick, D., U.S. Pat. No. 3,563,765 (1971)) treated dried egg yolk solids with hexane at a solvent to solid weight ratio of 3:1 at 80.degree. F. for 30 min. The amount of fat and cholesterol extracted by this process was 70% of the total content of each in the egg yolk solids. Merchent et al (Merchent, Z. M., et al., U.S. Pat. No. 5,037,661 (1991)) extracted egg yolk solids with isopropyl alcohol and hydrolyzed the cholesterol depleted-yolk with selected proteolytic enzymes to increase the interfacial tension coefficient, such that the resulting product was useful in producing emulsified products such as mayonnaise and salad dressings. Frioriti et al (Frioriti, et al., U.S. Pat. No. 4,103,040 (1978)) used safflower oil to decholesterolize egg yolk using an oil to wet yolk weight ratio of 4% and high shear mixing. The yolk was extracted three times and 80 to 83% of the cholesterol was removed.
Supercritical extraction (SCE) is used commercially for decaffeination of coffee and tea. Supercritical carbon dioxide extraction was investigated for removal of cholesterol and lipid components from dried yolk by Froning et al (Froning, G. W., et al., J. Food Sci. 55:95-98 (1990)). The maximum reduction obtained was 66% of the total cholesterol and about 50% of the fat. Recently, the Nutra-Sweet Company introduced a SCE process that removed 95% of egg yolk cholesterol and 80% lipid (Singer, N. S., et al., Paper No. 63, presented at the 53rd Annual Meeting of Inst. of Food technologists, Chicago, Illinois, August 8-15 (1993)). A major disadvantage of solvent use is that it extracts fat along with cholesterol, and may denature proteins.
Researchers are experimenting with an enzyme, cholesterol reductase, that converts cholesterol in the presence of NADPH to coprostanol, which passes through the body without being absorbed (Dehal, S. S., et al., A Novel Method to Decrease the Cholesterol Content of Foods, Fat and Cholesterol Reduced Foods (1991)). The enzyme is not commercially available due to the extremely high extraction costs.
Cholesterol was removed by adsorption or clathrazation with .beta.-cyclodextrin (.beta.-CD). In France, Bayol et al. (Bayol, A., et al., European Patent 0,326,469 (1989)) used .beta.-CD to reduce cholesterol in fresh egg yolk by 57%. Egg yolk was diluted 6 fold, mixed with .beta.-CD and shaken for 5 hours in a water bath. The complexes (.beta.-CD-cholesterol) were separated from the product by centrifugation. In the United States, a new reduced cholesterol egg product (80% cholesterol free) called "Simply Eggs" prepared using .beta.-CD, was recently introduced by Crystal Farms.
Cyclodextrins are commercially available cyclic oligosaccharides obtained by enzymatic degradation of starch. They consist of six, seven, or eight glucose monomers arranged in a donut shaped ring, which are denoted alpha, beta or gamma cyclodextrin, respectively. Cyclodextrins are water soluble due to the location of free hydroxyl groups on the external rim of the molecule. Solubility is a function of temperature. The higher the temperature the higher the solubility. The solubility of .beta.-cyclodextrins increases from 0.8% at 0.5.degree. C. to 39.7% at 90.degree. C. The internal cavity which is hydrophobic allows the cyclodextrins to complex molecules such as aromatic alcohols, fatty acids and their esters and cholesterol.
.beta.-cyclodextrin has been used to reduce cholesterol in egg yolk for several reasons: 1. The relative size and geometry of the .beta.-cyclodextrin internal cavity allowed good complexing with free and esterified cholesterol; 2. The realization of industrial scale production of .beta.-cyclodextrin; 3. The intensive research on toxicity of .beta.-cyclodextrin during the past decade, assured its safety as a food ingredient.
Six-month oral chronic toxicity of .beta.-cyclodextrin (Szejtli, J., Industrial applications of cyclodextrins. Inclusion Compound 3:331-390 (1984)) was studied in rats by feeding up to 1.6 g/body weight kg/day and up to 0.6 g/body weight kg/day in dogs. Weight gain, food consumption and clinico-biochemical values were not affected. ".beta.-cyclodextrin showed no embryo-toxic effect. Orally administered .beta.-cyclodextrin can thus be considered a non-toxic substance (Szejtli, J., Industrial applications of cyclodextrins. Inclusion Compound 3:331-375 (1984))". .beta.-cyclodextrin is allowed as a food ingredient in many countries, but not in the United States as yet.